Method of isolating lipids from a lipids containing biomass with aid of hydrophobic silica

ABSTRACT

The current invention relates to a method of isolating lipids from a lipids containing biomass with aid of hydrophobic silica and to an oil and to a delipidated biomass as obtained by such a method.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is US national stage of internationalapplication PCT/EP2019/061629, which had an international filing date ofMay 7, 2019, and which was published on Nov. 21, 2019. The PCTapplication claims priority to European application EP 18172317.2, filedon May 15, 2018. The contents of each of these applications is herebyincorporated by reference in its entirety.

The current invention relates to a method of isolating lipids from alipids containing biomass with aid of hydrophobic silica.

PUFAs (polyunsaturated fatty acids) containing lipids are of highinterest in the feed, food and pharmaceutical industry. Due tooverfishing there is a high need for alternative sources for PUFAscontaining lipids besides fish oil. It turned out that besides certainyeast and algal strains in particular microalgal cells like those of theorder Thraustochytriales are a very good source for PUFAs containinglipids.

But with respect to microbial organisms and in particular cells of theorder Thraustochytriales, which produce the PUFAs containing lipids, theisolation of the oil from the cells turned out as a particular problem.The most effective way of isolating the oil was the use of organicsolvents like hexane. But the use of organic solvents leads to hazardousoperating conditions, requires the use of expensive explosion-proofequipment and requires the implementation of an expensive solventrecovery process to avoid pollution of the environment.

In the attempt to avoid the use of organic solvents, as an effectivealternative way for isolating the oil has turned out the salting-out ofthe oil with high amounts of sodium chloride. But the use of highamounts of sodium chloride leads to a delipidated biomass by-productwhich due to the high salt content cannot be utilized as a feedingredient, so that the process is not very sustainable. Further, thehigh salt concentration leads to fast corrosion of the used steelequipment.

Thus, it was the object of the current invention to provide an effectivemethod for isolating a lipid, in particular a PUFAs containing lipid,from lipids containing cells, in particular of the orderThraustochytriales, and simultaneously avoiding not only the need oforganic solvents, but further avoiding the need of high amounts of saltsand preferably also avoiding the use of high amounts of base and/or ahigh pH for realizing the effective isolation of the oil from the cells.

It was a further object of the current invention to provide a method forisolating a lipid, in particular a PUFAs containing lipid, from lipidscontaining cells, in particular of the order Thraustochytriales, andsimultaneously to provide a delipidated biomass which can be utilized ina commercial way, preferably in the agricultural field.

It turned out that an effective isolation of the lipid from the biomasscan be realized, if the biomass, in particular after lysing, isincubated with hydrophobic silica, preferably at about neutral pH and ata temperature of between 50° C. and 100° C. By keeping the temperaturewell below 100° C. and avoiding high pH values, it was possible toprohibit at least essentially saponification of the fatty acid esters.Thus, an environmentally friendly, sustainable as well as mild methodfor isolating lipids from the cells of the biomass is provided.

As further there was no need to apply alkaline conditions and thus noalkaline agents and further also no organic solvents have to be added, abiomass was obtained which contained very low amounts of salts and/or isessentially free of organic solvents and by that turned out as veryuseful for applications in particular in the agricultural field.

Thus, a first subject of the current invention is a method of isolatinga lipid from a lipids containing biomass, comprising the followingsteps:

-   -   a) Providing a biomass suspension, wherein the cells of the        biomass contain in average at least 10 wt.-% of lipids;    -   b) Optionally lysing the cells of the biomass;    -   c) Adding hydrophobic silica to the suspension;    -   d) Heating the suspension thus obtained to a temperature of at        least 50° C., in particular 50 to 100° C., preferably 65 to 95°        C., and incubating the suspension for at least 20 minutes,        preferably 20 minutes to 30 hours, in particular 30 minutes to        26 hours, more preferably 1 hour to 24 hours, 2 to 20 hours, 3        to 15 hours or 4 to 10 hours;    -   e) Separating the oil containing light phase from the        cell-debris containing aqueous phase.

Preferably the steps are carried out in the order as depicted, butoptionally hydrophobic silica may be added before lysing of thesuspension or, on the other hand, hydrophobic silica may be added afterthe suspension has already been heated.

Preferably the biomass suspension is further concentrated before orafter addition of the hydrophobic silica to a total dry matter (TDM)content of 20 to 60 wt.-%, if it has a TDM content outside of thisrange.

Thus, a preferred subject of the current invention is a method ofisolating lipids from a lipids containing biomass, comprising thefollowing steps:

-   -   a) Providing a suspension of a biomass comprising cells which        contain a PUFAs containing lipid;    -   b) Optionally lysing the cells of the biomass;    -   c) Concentrating the suspension to a total dry matter (TDM)        content of 20 to 60 wt.-%, preferably 25 to 60 wt.-%, more        preferably 30 to 55 wt.-%, if the suspension has a lower TDM        content;    -   d) Adding hydrophobic silica to the suspension;    -   e) Heating the suspension thus obtained to a temperature of at        least 50° C., in particular 50 to 100° C., preferably 65 to 95°        C., and incubating the suspension for at least 20 minutes,        preferably 20 minutes to 30 hours, in particular 30 minutes to        26 hours, more preferably 1 hour to 24 hours, 2 to 20 hours, 3        to 15 hours or 4 to 10 hours;    -   f) Separating the oil set free by mechanical means from the        cell-debris containing aqueous phase.

In this embodiment of the invention steps (a) to (f) are preferablycarried out in the order as depicted. But optionally concentration ofthe biomass may also be carried out before lysing of the cells orhydrophobic silica may be added before lysing and/or concentrating ofthe suspension or, on the other hand, hydrophobic silica may be addedafter the suspension has already been heated.

The steps of lysing the cells, concentration of the suspension, additionof hydrophobic silica and incubation of the suspension with hydrophobicsilica are preferably carried out under mechanical mixing, preferably bystirring or agitating of the respective suspension.—Stirring and/oragitating may be carried out accordingly by using a stirrer and/or anagitator. In particular low shear agitation and/or axial-flow agitationmay be applied, in particular as disclosed in WO 2015/095694. Impellerssuitable for agitating include in particular straight blade impellers,Rushton blade impellers, axial flow impellers, radial flow impellers,concave blade disc impellers, high-efficiency impellers, propellers,paddles, turbines and combinations thereof.

In the methods according to the invention, the addition of silica leadsto the breaking of the emulsion as contained in the suspension into anoil containing light phase and a water, cell debris and salts containingheavy phase. This breaking of the emulsion is also called“demulsification” in the context of this application.

In a preferred embodiment of the invention, hydrophobic silica is addedto the suspension to adjust a final concentration in the suspension ofbetween 0.005 and 0.25 wt.-% of hydrophobic silica. In particular,hydrophobic silica is added in an amount to adjust a final concentrationof between 0.008 and 0.20 wt.-%, preferably 0.012 to 0.15 wt.-%, morepreferably 0.015 to 0.10 wt.-%.

According to the invention, “hydrophobic silica” may refer to a singlekind of hydrophobic silica or to a mixture of different kinds ofhydrophobic silicas.

Hydrophobic silicas are registered under CAS No. 68611-44-9 and arecommercially available, for example, under the trade names Sipernat® orAerosil® (Evonik Industries, Germany). Hydrophobic silicas arecharacterized by hydrophobic groups chemically bonded to the surface.The chemical groups are preferably alkyl or polydimethylsiloxane groups.Hydrophobic silica can be obtained, for example, by chemical treatmentof hydrophilic silica with alkoxy silanes, silazanes or siloxanes.

Hydrophobic silicas preferably used according to the invention have amethanol wettability of at least 40%, preferably at least 45%,particularly preferably at least 50%, particularly 40 to 65%, especially45 to 60%.

The methanol wettability is a measure of the hydrophobicity of thesilica powder. To determine this value, a certain amount of silicapowder is weighed into water. The silica powder remains here on thesurface. The amount of methanol required for wetting the powder is thendetermined. “Methanol wettability” is here understood to mean themethanol content of a methanol-water mixture in % by volume in which 50%of the hydrophobic silica sediments.

The hydrophobic silicas according to the invention preferably have atamped density (unsieved; based on ISO 787-11) of 100 to 200 g/l,preferably 125 to 175 g/l. The particle size of the hydrophobic silica(d50; laser diffraction; based on ISO 13320-1) is preferably from 2 to50 μm, more preferably from 5 to 20 μm, in particular from 8 to 15 μm.Loss on drying of the hydrophobic silica (2 hours at 105° C.; based onISO 787-2) is preferably at most 10%, particularly preferably at most6%. Ignition loss of the hydrophobic silica (2 hours at 1000° C.; basedon ISO 3262-1) is preferably at most 10%, particularly preferably atmost 6%.

The silicon dioxide content of the hydrophobic silica is preferably atleast 95% by weight, particularly preferably at least 97% by weight(based on ISO 3262-19). The carbon content of the hydrophobic silica ispreferably at most 3.5%, in particular 0.2 to 3.5%, by weight,particularly at most 2%, in particular 0.5 to 2%, by weight (based onISO 3262-19). The pH of the hydrophobic silica (5% in a 1:1 mixture ofwater and methanol; based on ISO 787-9) is preferably from 7 to 10.5,particularly preferably from 7.5 to 9.

Hydrophobic silica(s) which can be used in accordance with the inventionare, for example, obtainable under the trade names Sipernat® D 10,Sipernat® D13, Sipernat® D 15, Sipernat® D 17 and Aerosil® types R, RX,RY and NX, in particular Aerosil® R 202 (Evonik Industries, Germany).

In a preferred embodiment of the invention, the hydrophobic silica isused in form of a demulsifier mixture which contains besides thehydrophobic silica at least one surfactant. The at least one surfactantmay be selected from the group consisting of nonionic, anionic,zwitterionic, ampholytic and cationic surfactants. In a preferredembodiment of the invention the at least one surfactant is preferably anonionic surfactant.

Preferred anionic surfactants are alkyl sulfates, alkyl polglycol ethersulfates and ether carboxylic acids with 10 to 18 C atoms in the alkylgroup and up to 12 glycol ether groups in the molecule, sulfosuccinicacid mono and dialkyl esters with 8 to 18 C atoms in the alkyl group andsulfosuccinic acid mono-alkylpolyoxyethyl esters with 8 to 18 C atoms inthe alkyl group and 1 to 6 oxyethylene groups, monoglyceride sulfates,alkyl and alkenyl ether phosphates as well as condensates of protein andfatty acids.

Zwitterionic surfactants carry at least a quaternary ammonium group andat least one —COO<-→—or SO3<-→ group in the molecule. Particularlysuitable zwitterionic emulsifiers are the so-called betaines such asN-alkyl-N,N-dimethylammonium glycinates,N-acyl-aminopropyl-N,N-dimethylammonium glycinates and2-alkyl-3-carboxymethyl-3-hydroxyethyl-imidazolines, each having 8 to 18carbon atoms in the alkyl or acyl group, as well ascocoacylaminoethylhydroxyethylcarboxymethyl glycinate.

Exemplary nonionic surfactants usable according to the invention arelinear or branched C8-C30 fatty acids and their Na, K, ammonium, Ca, Mgand Zn salts; fatty acid alkanolamides; fatty acid glucamides;N-alkylglucamides; and C8-C22 alkylamine-N-oxides.

Preferred nonionic surfactants according to the invention arealkoxylated, in particular ethoxylated and/or propoxylated, preferablyethoxylated, compounds as well as alkylated sugars and in particularalkoxylated alkylated sugars.

Examples for alkoxylated compounds are alkoxylated fatty alcohols, sugaralcohols, fatty acids, alkylphenols, castor oil, hydrogenated castoroil, fatty acid alkanolamides and fatty acid amines; in particularaddition products of 4 to 30 moles ethylene oxide and/or 0 to 5 molespropylene oxide on linear C8-C22-fatty alcohols, on C12-C22-fatty acidsand on C8-C15-alkylphenols; C12-C22 fatty acid mono and diesters ofaddition products of 1 to 30 moles ethylene oxide on C3-C6 polyols,particularly on glycerin, ethylene oxide and polyglycerin; additionproducts on methylglucoside fatty acid esters, addition products of 5 to60 moles ethylene oxide on castor oil and hydrogenated castor oil,partial esters of polyols containing 3-6 carbon atoms with C8-C22 fattyacids; addition products of 1 to 30 moles ethylene oxide to fatty acidalkanolamides and fatty acid amines.

Further examples for alkoxylated compounds are alkoxylated fatty acidalkyl esters of the formula R<1>CO-(OCH2CHR<2>)xOR<3> in which R<1>COstands for a linear or branched, saturated and/or unsaturated acyl groupwith 6 to 22 carbon atoms, R<2> for hydrogen or methyl, R<3> for linearor branched alkyl groups with 1 to 4 carbon atoms and x for numbers from1 to 20.

Further examples of nonionic surfactants are alkyl mono-, oligo- andpolyglycosides corresponding to the general formula RO-(Z)x wherein Rstands for a C8-C22 alkyl, in particular C8-C16 alkyl, Z for sugar and xfor the number of sugar units. Any mono-, oligo- or polysaccharide canbe added as the sugar building block Z. Normally, sugars having 5 or 6carbon atoms as well as the corresponding oligosaccharides are added,for example, glucose, fructose, galactose, arabinose, ribose, xylose,lyxose, allose, altrose, mannose, gulose, idose, talose and/or sucrose.Preferred sugar building blocks are glucose, fructose and sucrose withglucose being particularly preferred, The inventively usable alkylpolyglycosides comprise an average of 1.1 to 5, preferably 1,1 to 2.0,particularly preferably 1.1 to 1.8 sugar units. The alkoxylated homologsof the cited alkyl polyglycosides can also be used according to theinvention. These homologs preferably comprise on average up to 10ethylene oxide and/or propylene oxide units per alkyl glycoside unit.

In a very preferred embodiment of the invention, fatty acid esters ofsugar alcohols and/or alkoxylated derivatives of such compounds are usedas nonionic surfactants. Preferred sugar alcohols in this context areglycerol, polyglycerol, sorbitol, isosorbide and sorbitans, inparticular 1,4-sorbitan. Examples of such fatty acid esters of sugaralcohols are polyglyceryl-2-dipolyhydroxy stearate andpolyglyceryl-3-diisostearate.

Very preferred examples of such fatty acid esters of sugar alcohols areesters of sorbitan, in particular of 1,4-sorbitan, with C12-C22 fattyacids, preferably with C16-C18 fatty acids, wherein the ester bond ispreferably formed with the omega hydroxyl group of the sorbitan.Particular examples are sorbitan monolaurate, sorbitan monopalmitate,sorbitan monostearate, sorbitan monooleate, sorbitan sesquioleate,sorbitan trioleate and sorbitan isostearate, with sorbitan monooleatebeing particularly preferred. Sorbitan monooleate is commerciallyavailable under the tradename Span® 80 (Croda, USA).

Very preferred examples of such alkoxylated fatty acid esters of sugaralcohols are esters of ethoxylated sorbitan, in particular ofethoxylated 1,4-sorbitan, with C12-C22 fatty acids, preferably withC16-C18 fatty acids, wherein preferably all free hydroxyl groups of thesorbitan do carry 1 to 5, preferably 1 to 3, ethoxylate groups andwherein the fatty acid bond is formed with the ethoxylated omegahydroxyl group of the sorbitan. These compounds are also calledpolysorbates. Particular examples are PEG-20 sorbitan monolaurate, PEG-4sorbitan monolaurate, PEG-20 sorbitan monopalmitate, PEG-20 sorbitanmonostearate, PEG-4 sorbitan monostearate. PEG-20 sorbitan tristearateand PEG-20 sorbitan monooleate, with PEG-20 sorbitan monooleate beingparticularly preferred. PEG-20 sorbitan monooleate, also known aspolysorbate 80, is commercially available under the tradename Tween® 80(Croda, USA),

In a preferred embodiment of the invention, a mixture of at least twononionic surfactants is used in the demulsifier composition. Preferablya mixture of a nonionic surfactant with a low HLB of preferably below 8,in particular from 3 to 6, and a nonionic surfactant with a high HLB ofpreferably above 12, in particular from 13 to 17, is used in thedemulsifier composition. Preferred examples for this mixture is amixture of Span® 80 and Tween® 80 (Croda, USA) or the mixture of Brij®92 and Brij® 98 (Uniqema, USA). (Concerning the definition of the HLBsee Römpp-Lexikon Chemie (Eds.: J. Falbe, M. Regitz), 10th edition,Georg Thieme Verlag Stuttgart, N.Y., (1997), page 1764).

Suitable emulsifiers with an HLB of below 8 are in particular compoundsof the general formula R<1>—O—R<2>, in which R<1> is a primary linearalkyl, alkenyl or acyl group having 18 to 30 carbon atoms, like thebehenyl, erucyl or arachidyl group, and R<2> is hydrogen, a group offormula -(CnH2nO)x-H with x=1 or 2 and n=2 to 4 or a polyhydroxyalkylgroup with 4 to 6 carbon atoms and 2 to 5 hydroxy groups, likepentaerythreitol, trimethylol propane, diglycerin or methylglucose.

In case that the hydrophobic silica is used as a demulsifier mixture incombination with at least one surfactant, then the ratio of hydrophobicsilica to surfactant preferably varies from 1:50 to 50:1. In a preferredembodiment of the invention, the hydrophobic silica is contained in thedemulsifier mixture in an amount of from 1 wt.-% to 50 wt.-%, preferablyin an amount of 2 to 25 wt.-%, in particular in an amount of 3 to 15wt.-%.

Preferably the surfactant(s) form(s) the residual part of thedemulsifier mixture, but besides surfactants further ingredients may beincluded in the demulsifier mixture, for example to modulate thephysical properties of the demulsifier mixture.

In a very preferred embodiment of the invention, a demulsifier mixtureis used, which comprises hydrophobic silica in an amount of 3 to 15wt.-% and at least one, preferably two, nonionic surfactant(s) in anamount of at least 85 wt.-%, wherein the nonionic surfactant(s) is/arepreferably selected from fatty acid esters of sugar alcohols and/oralkoxylated derivatives of such compounds, in particular from fatty acidesters of sorbitan, preferably sorbitan monooleate, and ethoxylatedfatty acid esters of sorbitan, preferably PEG-20 sorbitan monooleate,and mixtures thereof.

In another preferred embodiment of the invention the hydrophobic silicais used in combination with an oil, in particular in combination with atriglyceride containing oil, such as microalgal oil or vegetable oil,wherein examples for vegetable oils are soybean oil, canola oil and cornoil. If the hydrophobic silica is used in combination with an oil, thenthe ratio of hydrophobic silica to oil preferably varies from 1:50 to50:1, in particular from 1:1 to 1:20.

In a particular embodiment of the invention, the hydrophobic silica isemployed in combination with at least one nonionic surfactant andsimultaneously in combination with at least one oil, in particular incombination with at least one triglyceride containing oil.

As according to the invention, it is not necessary to raise or lower thepH value, the reaction is preferably carried out at about neutral pH toestablish mild conditions for avoiding saponification of the lipids.Thus, the pH is preferably kept between 5 and 9, more preferably between6 and 8, and very preferably between 6.5 and 8.0.—In case of afermentation broth, out of convenience the fermentation broth may beemployed, after the fermentation is ended, without any adjustment of thepH value.

If necessary or desired, the pH value may be adjusted by using bases oracids. In general, adjusting the pH value can be carried out accordingto the invention by using either bases or acids as known to thoseskilled in the art. Decreasing of the pH can be carried out inparticular by using organic or inorganic acids like sulfuric acid,nitric acid, phosphoric acid, boric acid, hydrochloric acid, hydrobromicacid, perchloric acid, hypochlorous acid, chlorous acid, fluorosulfuricacid, hexafluorophosphoric acid, acetic acid, citric acid, formic acid,or combinations thereof. As a high content of chloride is desirablyavoided, in a preferred embodiment of the invention no or only smallamounts of hydrochloric acid are used in the process of the currentinvention. According to the invention, sulfuric acid is the preferredsubstance for decreasing the pH value.—Increasing of the pH can becarried out in particular by using organic or inorganic bases likehydroxides, in particular sodium hydroxide, lithium hydroxide, potassiumhydroxide, and/or calcium hydroxide, carbonates, in particular sodiumcarbonate, potassium carbonate, or magnesium carbonate, and/orbicarbonates, in particular lithium bicarbonate, sodium bicarbonate,and/or potassium bicarbonate.—Due to easiness of handling, the acids andbases are preferably used in liquid form, in particular as concentratedsolutions, wherein the concentration of acid or base in the solution ispreferably in the range of 10 to 55 wt.-%, in particular in the range of20 to 50 wt.-%. In particular sulfuric acid is preferably used also inconcentrated form.

According to the invention after providing the suspension according tostep (a) preferably a lysing step is carried out. The lysing step can beomitted, if—for example due to the fermentation conditions asapplied—the cells or a big part thereof is already lysed or easilybreakable in one of the following steps of the procedure without anyexplicit lysing step.

Lysing of the cells of the biomass according to step (b) can be carriedout by methods as known to those skilled in the art, in particularenzymatically, mechanically, physically, or chemically, or by applyingcombinations thereof.

Depending on the time of exposure and/or the degree of force applied, acomposition comprising only lysed cells or a composition comprising amixture of cell debris and intact cells may be obtained. The term “lysedlipids containing biomass” insofar relates to a suspension whichcontains water, cell debris and oil as set free by the cells of thebiomass, but beyond that may also comprise further components, inparticular salts, intact cells, further contents of the lysed cells aswell as components of a fermentation medium, in particular nutrients. Ina preferred embodiment of the invention, only small amounts of intactcells, in particular less than 20%, preferably less than 10%, morepreferably less than 5% (relating to the total number of intact cells aspresent before lysing the cells of the biomass) are present in the lysedbiomass after the step of lysing the cells.

Lysing of the cells may be realized for example by utilizing a Frenchcell press, sonicator, homogenizer, microfluidizer, ball mill, rod mill,pebble mill, bead mill, high pressure grinding roll, vertical shaftimpactor, industrial blender, high shear mixer, paddle mixer, and/orpolytron homogenizer.

In a preferred embodiment of the invention, lysing of the cellscomprises an enzymatic treatment of the cells by applying a cell-walldegrading enzyme.

According to the invention, the cell-wall degrading enzyme is preferablyselected from proteases, cellulases (e.g., Cellustar CL (Dyadic),Fibrezyme G2000 (Dyadic), Celluclast (Novozymes), Fungamyl (Novozymes),Viscozyme L (Novozymes)), hemicellulases, chitinases, pectinases (e.g.,Pectinex (Novozymes)), sucrases, maltases, lactases, alpha-glucosidases,beta-glucosidases, amylases (e.g., Alphastar Plus (Dyadic); Termamyl(Novozymes)), lysozymes, neuraminidases, galactosidases,alpha-mannosidases, glucuronidases, hyaluronidases, pullulanases,glucocerebrosidases, galactosylceramidases, acetylgalactosaminidases,fucosidases, hexosaminidases, iduronidases, maltases-glucoamylases,xylanases (e.g., Xylanase Plus (Dyadic), Pentopan (Novozymes)),beta-glucanases (e.g., Vinoflow Max (Novozymes), Brewzyme LP (Dyadic)),mannanases, and combinations thereof. The protease may be selected fromserine proteases, threonine proteases, cysteine proteases, aspartateproteases, metalloproteases, glutamic acid proteases, alcalases(subtilisins), and combinations thereof. The chitinase may be achitotriosidase. The pectinase may be selected from pectolyases,pectozymes, polygalacturonases, and combinations thereof.

The adequate pH for utilizing the enzyme depends on the pH optimum ofthe enzyme.

In a preferred embodiment of the invention, an enzyme with a pH optimumof between 7.0 and 9.0, in particular of about 7.5, is used, so that thepH applied in this step is from 7.0 to 9.0, in particular from 7.0 to8.0, preferably from 7.3 to 7.7. A preferred enzyme which can be used inthis pH range is an alcalase.

The enzyme is preferably added as a concentrated enzyme solution,preferably in an amount of 0.01 to 1.5 wt.-%, more preferably in anamount of 0.03 to 1.0 wt.-%, above all in an amount of 0.05 to 0.5wt.-%, relating to the amount of concentrated enzyme solution as addedin relation to the total amount of the suspension after addition of theconcentrated enzyme solution.

In a very preferred embodiment of the invention, lysing of the cells iscarried out as follows:

-   -   i) Heating the suspension of (a) to a temperature of between        50° C. and 70° C., preferably to a temperature of between 55° C.        and 65° C., and adding a cell wall-degrading enzyme to the        suspension, in particular fermentation broth, and adjusting an        adequate pH value, if necessary, at which the enzyme is properly        working;    -   ii) Keeping the temperature and pH in the ranges as depicted        in (ii) for at least one hour, preferably for at least two        hours, more preferably for two to four hours.

In step (i), the enzyme can be added before or after heating up thesuspension and/or before or after adjusting the pH. In the same wayheating up of the suspension can be carried out before or afteradjusting the pH.—But in a preferred embodiment, the enzyme is addedafter heating up of the suspension and after adjusting the pH, ifadjusting of the pH is necessary, at all.—In a very preferred embodimentall measures are carried out more or less simultaneously.

Preferably, in the steps (i) and (ii) the suspension is continuouslymixed by using a stirrer and/or an agitator.

According to the invention, the demulsification is preferably carriedout with a suspension having a dry matter content of 20 to 60 wt.-%,preferably 25 to 60 wt. %, in particular 30 to 55 wt.-% or 30 to 45wt.-%. This can be realized by either providing a suspension with anappropriately high biomass in step (a) or by concentrating thesuspension, in particular after lysing the cells of the biomass. Thus,in a preferred embodiment of the invention, after optional lysing of thecells of the biomass and before the demulsification step, the suspensionis concentrated to a total dry matter content of 20 to 60 wt.-%, morepreferably 25 to 60 wt.-%, in particular 30 to 55 wt.-%, above all 30 to50 wt.-% or 30 to 45 wt.-%.

Concentration of the suspension is preferably carried out by evaporationof water at a temperature not higher than 100° C., preferably 70° C. to100° C., more preferably 80° C. to 90° C., until a total dry mattercontent of 20 to 60 wt.-% more preferably 25 to 60 wt.-%, in particular30 to 55 wt.-% or 30 to 45 wt.-%, is reached.

Concentration of the suspension is preferably carried out in a forcedcirculation evaporator (for example available from GEA, Germany) toallow fast removal of the water. Alternatively or in addition,concentration might be carried out by falling film evaporation, thinfilm evaporation and/or rotary evaporation.

The method according to the invention comprises as a further step theharvesting of the PUFAs containing lipid from the demulsifiedcomposition by separation of the oil containing light phase from thewater, salts, cell debris and residual oil containing aqueous phase.

The harvesting of the PUFAs containing lipid may comprise as an optionalstep the neutralization of the demulsified suspension, if thedemulsification was not carried out at neutral pH.

Neutralization of the demulsified composition is preferably realized, ifnecessary or desired, by adding an acid, preferably sulfuric acid, orbase, preferably caustic soda, to adjust a pH value of 5 to 9,preferably 5.5 to 8.5, in particular 6.0 to 8.0 or 6.5 to 7.5. Beforestarting separation of the light phase from the aqueous phase, theneutralized composition may be stirred at this neutralized pH fromseveral minutes up to several hours.

Separation of the oil containing light phase from the water, salts andcell debris containing heavy phase is preferably realized by mechanicalmeans and preferably at a temperature of 60-90° C., more preferably70-80° C., and preferably at a pH value of 6-9, more preferably 7-8.5.“Mechanical means” refers in particular to filtration and centrifugationmethods as known to those skilled in the art.

After separation of the oil containing light phase, the PUFAs containingoil thus obtained can further be worked up by applying methods as knownto those skilled in the art, in particular refining, bleaching,deodorizing and/or winterizing.

A particular advantage of the method of the current invention is that itcan be carried out without the use of any organic solvent, in particularwithout the use of any polar or non-polar organic solvent. Thus, in apreferred embodiment of the invention, no or only little amounts oforganic solvents, in particular of polar or non-polar organic solvents,are used for isolating the PUFAs containing oil from the biomass.Typical organic solvents are hexane and ethanol. In a preferredembodiment of the invention less than 2 wt.-% non-polar organic solventsare used, more preferably less than 1, 0.5 or 0.1 wt.-%. In aparticularly preferred embodiment of the invention no non-polar organicsolvent is used, at all. In a very preferred embodiment of the inventionless than 2 wt.-% organic solvents are used, in general, particularlypreferred less than 1, 0.5 or 0.1 wt.-%. In a particularly verypreferred embodiment of the invention no organic solvents are used, atall, for isolating the PUFAs containing oil from the biomass.—This meansin particular for this embodiment that the suspension as employed in themethod according to the invention as well as all compositions asobtained by said single method steps preferably contain non-polarorganic solvents, preferably organic solvents in general, in an amountof less than 2 wt.-%, more preferably less than 1 wt.-%, in particularless than 0.5 or 0.3 wt.-%, above all in an amount of less than 0.1 or0.05 wt.-%.

A further advantage of the method of the current invention is that avery effective separation of the oil from the remaining biomass can berealized without the addition of sodium chloride, which is normally usedfor salting out the oil from the biomass. Preferably the method can becarried out without the addition of chloride salts, at all, above allwithout the addition of any salts for salting out the oil. But smallamounts of chloride salts, in particular sodium chloride, might bepresent in the suspension due to the fermentation medium as used forgrowing of the biomass.

Thus, in a preferred embodiment of the current invention, no or onlylittle amounts of sodium chloride are used for improving the oilisolation. In a preferred embodiment of the invention less than 1 wt.-%of sodium chloride, are used, more preferably less than 0.5 or 0.2 wt.-%of sodium chloride are used for isolating the oil from the biomass,above all less than 0.1 or 0.05 wt.-%, wherein the wt.-% relate to thetotal weight of the composition after addition of the sodiumchloride.—This means in particular for this embodiment that thesuspension as employed in the method according to the invention as wellas all compositions as obtained by said single method steps preferablycontain sodium chloride in an amount of less than 2 wt.-%, morepreferably less than 1 wt.-%, in particular less than 0.5 or 0.3 wt.-%,above all in an amount of less than 0.1 or 0.05 wt.-%.

In a particularly preferred embodiment of the invention no or onlylittle amounts of chloride salts are used for improving the oilisolation, at all. In this embodiment preferably less than 1 wt.-% ofchloride salts, more preferably less than 0.5 or 0.2 wt.-% of chloridesalts are used for isolating the oil from the biomass, above all lessthan 0.1 or 0.05 wt.-%, wherein the wt.-% relate to the total weight ofthe composition after addition of the chloride salts.—This means inparticular for this embodiment that the suspension as employed in themethod according to the invention as well as all compositions asobtained by said single method steps preferably contain chloride, inparticular chloride salts, in an amount of less than 2 wt.-%, morepreferably less than 1 wt.-%, in particular less than 0.5 or 0.3 wt.-%,above all in an amount of less than 0.1 or 0.05 wt.-%.

In a very preferred embodiment of the invention no or only littleamounts of salts are used for improving the oil isolation, in general.In this embodiment preferably less than 1 wt.-% of salts, morepreferably less than 0.5 or 0.2 wt.-% of salts are used for isolatingthe oil from the biomass, above all less than 0.1 or 0.05 wt.-%, whereinthe wt.-% relate to the total weight of the composition after additionof the salts.—This means that preferably, in particular for thisembodiment of the invention, that the suspension as employed in themethod according to the invention as well as all compositions asobtained by said single method steps preferably contain salts in generalin an amount of less than 2 wt.-%, more preferably less than 1 wt.-%, inparticular less than 0.5 or 0.3 wt.-%, above all in an amount of lessthan 0.1 or 0.05 wt.-%.

A further advantage of the method of the current invention is that avery efficient separation of the oil from the remaining biomass can berealized without the addition of bases like caustic soda, which arenormally used for breaking the emulsion. Preferably the method can becarried out without the addition of bases, at all, so that also nosubsequent neutralization of the suspension is needed.—This does resultin a biomass which contains only relatively small amounts of salts, ingeneral, and by that comprises only a small amount of ashes. But smallamounts of bases might be added, in particular for adjusting the pH, forexample if the pH of the suspension is too low after the fermentation,or to facilitate the demulsification process.—That means that in apreferred embodiment of the invention no or only little amounts of basesare used in the methods according to the invention. In a preferredembodiment of the invention less than 4 wt.-% of bases, are used, morepreferably less than 2 or 1 wt.-% of bases are used in the methodsaccording to the invention, above all less than 0.5 or 0.2 wt.-%,wherein the wt.-% relate to the total weight of the composition afteraddition of the base.

But in an alternative embodiment of the invention caustics, inparticular caustic soda, may be added, preferably in small amounts, tofacilitate the demulsification and to increase the final oil yield.

As the demulsification can be carried out without the use of bases, in apreferred embodiment of the invention, the pH can preferably kept duringthe complete procedure below a pH value of 9, in particular below 8.5, 8or 7.5, which allows treatment of the lipid under very mild conditionsand substantially reduces the degree of hydrolysis of the fatty acidesters as contained in the lipid.—Thus, in a preferred embodiment of theinvention, the pH is kept during the complete procedure below 9, inparticular in the range of 4.5 to 9, preferably below 8.5, 8 or 7.5, inparticular in the range of 4.5 to 8.5, 4.5 to 8 or 4.5 to 7.5.

The methods of the current invention allow a very efficient separationof the oil contained in the biomass from the cell-debris and othersubstances as contained in the suspension, in particular fermentationbroth. By using the methods of the current invention preferably morethan 80 wt.-%, in particular more than 90 wt.-% of the oil contained inthe biomass can be separated from the biomass and isolated by applyingvery economic and sustainable conditions.

The yield of liberated oil is preferably determined by fatty acid methylester analysis (FAME). For doing that the lipids in the sample are firstsaponified with KOH. After that, the free fatty acids are methylatedwith MeOH. The methylated fatty acids can then be determined andquantified by gas chromatography by using an internal standard.

It turned out further that the oil as obtained by applying the method ofthe current invention has some advantageous characteristics over thePUFAs containing oils as disclosed in the state of the art so far. Inparticular it exhibits very low oxidation values and a very low contentof free fatty acids. Further, due to the method of preparation, the oilcontains a certain amount of hydrophobic silica.

Thus, a further subject of the current invention is an oil as obtainedor as obtainable by a method according to the current invention.

A further subject of the current invention is therefore also an oil,preferably a PUFAs containing oil, comprising hydrophobic silica in anamount of 5 ppm (w/w) to 10 wt.-%, preferably 10 ppm (w/w) to 5 wt.-%,in particular 0.01 wt.-% to 4 wt.-%, 0.1 wt.-% to 3 wt.-% or 0.5 wt.-%to 2.5 wt.-%.

In a very preferred embodiment only small amounts of hydrophobic silicaare used for breaking the emulsion, so that a particular embodiment ofthe invention is a PUFAs containing oil, comprising hydrophobic silicain an amount of 5 to 200 ppm (w/w), preferably 10 to 150 ppm (w/w), morepreferably 20 to 100 ppm (w/w), in particular 20 to 80 ppm (w/w).

The oil according to the invention preferably comprises PUFAs in anamount of at least 10 wt.-%, preferably at least 20 wt.-%, in particular25 to 70 wt.-%, 30 to 65 wt.-% or 45 to 60 wt.-%.

The oil according to the invention preferably comprises free fatty acids(FFA) in an amount of less than 1 wt.-%, preferably less than 0.8 wt.-%,more preferably less than 0.5 wt.-%. Free fatty acids are undesirableby-products in the isolation of TAGs containing lipids, which preferablyare avoided.

A further subject of the current invention is therefore also an oil, inparticular a PUFAs containing oil, which comprises fatty acid esters, inparticular TAGs, in an amount of at least 50 wt.-%, preferably at least70 or 80 wt.-%, in particular in an amount of at least 85 or 90 wt.-%,characterized by comprising free fatty acids (FFAs) in an amount of lessthan 0.6 wt.-%, preferably in an amount of less than 1 wt.-%, inparticular in an amount of less than 0.8 wt.-%, more preferably in anamount of less than 0.5 wt.-%.

A further subject of the current invention is therefore also a PUFAscontaining oil exhibiting the following characteristics: a) hydrophobicsilica in an amount of 5 ppm to 10 wt.-%, preferably 10 ppm to 5 wt.-%,in particular 0.01 wt.-% to 4 wt.-%, 0.1 wt.-% to 3 wt.-% or 0.5 wt.-%to 2.5 wt.-%; b) preferably a content of free fatty acids of less than 1wt.-%, preferably less than 0.8 wt.-%, in particular less than 0.5wt.-%; c) preferably a peroxide value of less than 0.5, more preferablyless than 0.3, in particular less than 0.15; d) preferably an anisidinevalue of less than 15, more preferably less than 10; e) preferably acontent of moisture and impurities of less than 1 wt.-%, preferably lessthan 0.5 wt.-%; f) preferably a viscosity of less than 250 cps, morepreferably of less than 200 cps, in particular of less than 160 cps; g)preferably a flash point of at least 350° C., more preferably of atleast 400° C., in particular of at least 450° C.; h) preferably acontent of omega-3 fatty acids, in particular of DHA and EPA, of atleast 35 wt.-%, preferably at least 40 or 45 wt.-%, above all at least50 wt.-%; i) preferably DHA and EPA each in an amount of at least 8wt.-%, preferably at least 10 wt.-% above all at least 15 wt.-%; j)preferably a content of triacylglycerols of at least 50 wt.-%, inparticular at least 70 or 80 wt.-%, more preferably at least 90 wt.-%;k) preferably an amount of organic solvents of less than 0.5 wt.-%, morepreferably less than 0.1 wt.-%, in particular less than 0.05 wt.-%,above all less than 0.01 wt.-%; l) preferably an amount of chlorides ofless than 0.1 wt.-%, more preferably less than 0.05 wt.-%, in particularless than 0.01 wt.-%; m) preferably a content of crude fat of more than90 wt.-%.

The anisidine value (AV) is determined in accordance with AOCS OfficialMethod Cd 18-90. The AV is a measure for secondary reaction products ofthe fatty acids, such as aldehydes and ketones, that occur duringoxidation of the oil.

The peroxide value (PV) is determined in accordance with the AOCSOfficial Method CD 8-53. The PV is a measure for primary reactionproducts, such as peroxide and hydroperoxides, that occur duringoxidation of the oil.—According to the invention the PV is measured inmeq/kg.

The content of free fatty acids is determined in accordance with AOCSOfficial Method AOCS Ca 5a-40. The content of moisture is determined inaccordance with AOCS Official Methods AOAC 930.15, 935.29. The contentof insoluble impurities is determined in accordance with AOCS OfficialMethod AOCS 3a-46. The amount of DHA and EPA is determined in accordancewith AOCS Official Method AOCS Ce 1b-89. The amount of total fat isdetermined in accordance with AOCS Official Method AOCS 996.06. Theamount of crude fat is determined in accordance with AOCS OfficialMethods AOAC 920.39, 954.02.

As the isolation of the oil is carried out by using no or only smallamounts of solvents and by also using no or only small amounts of sodiumchloride, the aqueous phase obtained as a by-product is preferablysubstantially free of organic solvents and sodium chloride, as well.Thus, the aqueous phase can be utilized in different ways, eitherdirectly after separation of the oil phase or after further work-up likeconcentrating and/or drying.

A further subject of the current invention is therefore a lipids, inparticular PUFAs, containing aqueous suspension, containing a biomass,preferably a delipidated biomass, as obtained or as obtainable by amethod according to the current invention. A further subject of thecurrent invention is therefore also a concentrate or a dried product asobtained or obtainable by concentrating and/or drying this aqueoussuspension. When concentrating the aqueous suspension, it is preferablydried until a total dry matter (TDM) content of 20 to 60 wt.-% isreached.—In the following the expression “aqueous suspension accordingto the invention” refers to the aqueous phase as obtained afterseparation of the oil phase as well as to any concentrated suspensionsof this aqueous phase as obtained by concentrating this aqueous phase.Drying is preferably carried out by solvent evaporation, as describedfurther below.

A further subject of the current invention is therefore also a lipids,in particular PUFAs, containing aqueous suspension, containing abiomass, in particular cell debris of a delipidated biomass,characterized by a content of salts of less than 15 wt.-%, preferably 4to 12 wt.-%, in particular 6 to 10 wt.-%, and preferably characterizedfurther by a content of non-polar organic solvents of less than 1 wt.-%,preferably less than 0.5 or 0.2 wt.-%, more preferably less than 0.1 or0.05 wt.-%, above all less than 0.01 wt.-%, and preferably furthercharacterized by a content of chloride ions of less than 1 wt.-%,preferably less than 0.5 or 0.2 wt.-%, more preferably less than 0.1 or0.05 wt.-%.

A further subject of the current invention is therefore in particularalso a lipids, in particular PUFAs, containing aqueous suspension,containing a biomass, in particular, cell debris of a delipidatedbiomass, characterized by a content of salts of less than 15 wt.-%,preferably 4 to 12 wt.-%, in particular 6 to 10 wt.-%, and preferablycharacterized further by a content of organic solvents of less than 1wt.-%, preferably less than 0.5 or 0.2 wt.-%, more preferably less than0.1 or 0.05 wt.-%, above all less than 0.01 wt.-%, and preferablyfurther characterized by a content of chloride ions of less than 1wt.-%, preferably less than 0.5 or 0.2 wt.-%, more preferably less than0.1 or 0.05 wt.-%.

A preferred subject of the current invention is therefore also a lipids,in particular PUFAs, containing aqueous suspension, containing aThraustochytrid biomass, in particular cell debris of a delipidatedThraustochytrid biomass, characterized by a content of salts of lessthan 15 wt.-%, preferably 4 to 12 wt.-% in particular 6 to 10 wt.-%, andpreferably characterized by a content of non-polar organic solvents ofless than 1 wt.-%, preferably less than 0.5 or 0.2 wt.-%, morepreferably less than 0.1 or 0.05 wt.-%, above all less than 0.01 wt.-%,and preferably further characterized by a content of chloride ions ofless than 1 wt.-%, preferably less than 0.5 or 0.2 wt.-%, morepreferably less than 0.1 or 0.05 wt.-%.

A particularly preferred subject of the current invention is thereforealso a lipids, in particular PUFAs, containing aqueous suspension,containing a Thraustochytrid biomass, in particular, cell debris of adelipidated Thraustochytrid biomass, characterized by a content of saltsof less than 15 wt.-%, preferably 4 to 12 wt.-%, in particular 6 to 10wt.-%, and preferably characterized by a content of organic solvents ofless than 1 wt.-%, preferably less than 0.5 or 0.2 wt.-%, morepreferably less than 0.1 or 0.05 wt.-%, above all less than 0.01 wt.-%,and preferably further characterized by a content of chloride ions ofless than 1 wt.-%, preferably less than 0.5 or 0.2 wt.-%, morepreferably less than 0.1 or 0.05 wt.-%.

The aqueous suspensions of the invention as described before preferablyexhibit a total dry matter (TDM) content of 20 to 60 wt.-%, inparticular of 25 to 55 wt.-%, more preferably of 30 to 50 wt.-%, as suchconcentrated suspensions turned out as particularly suitable for theapplications of the invention as described below.

The aqueous suspensions of the invention can be used as a product fordifferent purposes as disclosed further below.—Alternatively the aqueoussuspension may also be worked up further to increase the overall oilyield. The further work-up and isolation of further oil may be carriedout by using caustic soda.

“Chloride” according to the invention refers to the amount of detectablechlorine. The amount of chlorine as present can be determined forexample by elemental analysis according to DIN EN ISO 11885. Thechlorine is present in the form of salts which are called “chlorides”.The content of chloride as mentioned according to the invention—alsocalled “chloride ions”—only refers to the amount of detectable chlorine,not to the amount of the complete chloride salt, which comprises besidesthe chloride ion also a cationic counterion.

The total dry matter content (TDM) is preferably determined bygravimetric analysis. For doing that, a sample of the homogeneoussuspension with a specific volume is weighed before and afterfreeze-drying. The remaining weight of the dried sample corresponds tothe total dry matter as contained in that specific volume of thesuspension.

In a particularly preferred embodiment of the current invention thewater, salts, residual oil and cell debris containing aqueous phase,which is obtained as by-product in the oil harvesting step as describedbefore, is converted into a dried biomass by drying the biomass to atotal dry matter content of more than 90 wt.-%.

Due to the method of manufacture, the biomass comprises a very lowcontent of salts, preferably ashes, of less than 30 wt.-%, in particular15 to 20 wt.-%.

Thus, a further subject of the current invention is also a lipids,preferably PUFAs, containing biomass, in particular a delipidatedbiomass, characterized by a content of ashes, in particular salts, ofless than 30 wt.-%, preferably 8 to 30 wt.-%, more preferably 12 to 20wt.-%, in particular 15 to 20 wt.-%.

The biomass, in particular delipidated biomass, according to theinventon is preferably characterized by a content of non-polar organicsolvents of less than 2 wt.-%, preferably less than 1, 0.5 or 0.2 wt.-%,more preferably less than 0.1, 0.05 or 0.02 wt.-% and preferably furthercharacterized by a content of chloride ions of less than 2 wt.-%,preferably less than 1, 0.5 or 0.2 wt.-%, more preferably less than 0.1or 0.05 wt.-%.

Thus, a preferred subject of the current invention is also a lipids, inparticular PUFAs, containing Thraustocyhtrid biomass, in particular adelipidated Thraustochytrid biomass, characterized by a content ofashes, in particular salts, of less than 30 wt.-%, preferably 8 to 30wt.-%, more preferably 12 to 20 wt.-%, in particular 15 to 20 wt.-%, andpreferably a content of non-polar organic solvents of less than 2 wt.-%,preferably less than 1, 0.5 or 0.2 wt.-%, more preferably less than 0.1,0.05 or 0.02 wt.-% and preferably further characterized by a content ofchloride ions of less than 2 wt.-%, preferably less than 1, 0.5 or 0.2wt.-%, more preferably less than 0.1 or 0.05 wt.-%.

Thus, a particularly preferred subject of the current invention is alsoa lipids, in particular PUFAs, containing Thraustocyhtrid biomass, inparticular a delipidated Thraustochytrid biomass, characterized by acontent of ashes, in particular salts, of less than 30 wt.-%, preferably8 to 30 wt.-%, more preferably 12 to 20 wt.-%, in particular 15 to 20wt.-%, and preferably a content of organic solvents of less than 2wt.-%, preferably less than 1, 0.5 or 0.2 wt.-%, more preferably lessthan 0.1, 0.05 or 0.02 wt.-% and preferably further characterized by acontent of chloride ions of less than 2 wt.-%, preferably less than 1,0.5 or 0.2 wt.-%, more preferably less than 0.1 or 0.05 wt.-%.

As preferably the preparation is carried out without the use ofnon-polar organic solvents, preferably without the use of any organicsolvents, at all, and without the use of sodium chloride, preferablywithout the use of chloride salts, at all, the resulting biomass ispreferably free of any non-polar organic solvents, preferably free ofany organic solvents, in general, and further essentially free of anychloride ions, at all, wherein “essentially free” means that it containschloride ions in an amount of less than 0.1 wt.-%, in particular in anamount of less than 0.05 wt.-%.

The biomass according to the invention exhibits preferably a moisturecontent of not more than 10 wt.-%, preferably not more than 5 wt.-%.

The biomass thus obtained preferably comprises lipids (crude fat) in anamount of about 3 to 14 wt.-%, in particular about 4 to about 14 wt.-%,preferably in an amount of about 4.5 to about 12 wt.-%, more preferablyin an amount of about 5 to about 10 wt.-%. Further, the lipid preferablycomprises at least one PUFA selected from DHA and EPA, more preferably amixture of DHA and EPA, wherein the ratio of DHA to EPA is preferablybetween 3:2 to 4:1 and wherein the amount of DHA is preferably from 30to 50 wt.-% of the total amount of lipids contained and the amount ofEPA is preferably from 10 to 20 wt.-%. of the total amount of lipidscontained. Accordingly, also the aqueous suspensions as described beforeare preferably characterized by being convertible by drying into abiomass with such a crude fat content and/or EPA content and/or DHAcontent by drying the aqueous suspension to a moisture content of notmore than 10 wt.-%, preferably not more than 5 wt.-%.

The biomass preferably further comprises proteins and/or amino acids inan amount of 15 to 25 wt.-%, more preferably in an amount of 17 to 23wt.-%, and exhibits preferably a crude protein content of 25 to 35wt.-%. As the demulsification is carried out under mild conditions, theweight ratio of free amino acids to the sum of proteins and peptides ispreferably less than 9:1, more preferably less than 5:1, in particularless than 1:1. Accordingly, also the aqueous suspensions as describedbefore are preferably characterized by being convertible by drying intoa biomass with such an amino acid and/or crude protein content and/orration of free amino acids to proteins and peptides by drying theaqueous suspension to a moisture content of not more than 10 wt.-%,preferably not more than 5 wt.-%.

The biomass preferably further exhibits a crude fiber content of lessthan 5 wt.-%, preferably less than 2 wt.-%, more preferably of about 0wt.-%. Accordingly, also the aqueous suspensions as described before arepreferably characterized by being convertible by drying into a biomasswith such a crude fiber content by drying the aqueous suspension to amoisture content of not more than 10 wt.-%, preferably not more than 5wt.-%.

The dried biomass is preferably a delipidated biomass, that means abiomass, of which the major part of the lipids have been removed,preferably by a process as disclosed in this application. As theseparation of oil from the biomass is very effectively, the remainingoil in the biomass is preferably less than 20 wt.-%, preferably lessthan 15 wt.-%, more preferably less than 10 wt.-%, of the oil asoriginally contained in the biomass. But as the oil cannot be removedcompletely by such a process, a substantial amount of oil is stillcontained also in the delipidated biomass according to the invention.That means that the term “delipidated biomass” according to theinvention refers to a lysed biomass, from which the major part of oilhas been removed, preferably by a process or method as disclosed in thisapplication, but which still contains a substantial part of lipids, inparticular of PUFAs containing lipids, wherein the amount of lipids inthe dried delipidated biomass is preferably from 3-14 wt.-%, inparticular 4-14 wt.-%, preferably from 4.5-12 wt.-%, more preferablyfrom 5-10 wt.-%. Thus, the “delipidated biomass” according to theinvention might also be called a “partially delipidated biomass” or a“substantially delipidated biomass”.

Thus, a further subject of the current invention is a method ofobtaining a biomass which has a low content of salts, is substantiallyfree of non-polar organic solvents, preferably free of organic solvents,in general, and which is further substantially free of sodium chloride,preferably free of chloride salts, in general, comprising the methodsteps as mentioned before.

Conversion of the water, salts, remaining oil and cell debris containingheavy phase, which is obtained as by-product in the oil harvesting step,into a dried biomass by drying the biomass to a total dry matter contentof more than 90 wt.-%, can be carried out in different ways.

In a very preferred way, the transformation is carried out byconcentration of the heavy phase to a dry matter content of 30-50 wt.-%,preferably 35-45 wt.-%, and subsequent spray granulation of the biomassby means of fluidized bed granulation. By doing that, in a veryefficient way, a biomass with advantageous features can be obtained.Spray granulation by means of fluidized bed granulation is disclosed inmore detail in EP13176661.0.

Concentration of the heavy phase to a dry matter content of 30-50 wt.-%is preferably carried out by solvent evaporation, in particular vacuumevaporation, and/or by using a rotary evaporator, a thin-film evaporatoror a falling-film evaporator. A useful alternative to solventevaporation is reverse osmosis.

As alternative to the spray-granulation other drying methods, inparticular other convective drying methods, like tunnel drying or spraydrying, in particular nozzle spray drying, or contact drying methods,like drum drying, or radiation drying methods, like infrared drying, ofthe concentrated heavy phase would be applicable alternatives, whereinby using those methods normally particles with a smaller or biggerdiameter are obtained.

According to the invention, during the drying process, an anti-cakingagent, in particular silica, preferably a hydrophobic or hydrophilicsilica, may optionally be added to the biomass to prevent caking. Forthis purpose, the suspension, in particular fermentation broth,comprising biomass as well as the silica are preferably sprayed into theparticular drying zone. Alternatively or additionally, the biomass maybe mixed with the anti-caking agent after the drying process. Withrespect to the use of silica as anti-caking agent reference is made inparticular to the patent application EP13187631.0.

Conversion of a fine-grained powder into a coarse-grained dust-freeproduct can be realized by granulating processes. Conventional organicor inorganic auxiliaries or supports such as starch, gelatin, cellulosederivatives or similar substances, which are typically used in foodprocessing or feed processing as binding agents, gelling agents orthickeners, may optionally be used in this subsequent granulationprocess. Further auxiliaries that are preferably used according to theinvention are disclosed in WO 2016/050560, with carboxymethylcellulosebeing a particulary preferred binding agent.

After drying and optionally granulating and/or sieving of the biomass,the dried biomass is preferably stored or packed.

The particulate biomass of the invention as well as the aqueoussuspensions of the invention can be used in different ways. For example,they can be used in order to produce a foodstuff or feedstuff.Alternatively they may be used directly as foodstuff or feedstuff.

A further subject matter of the present invention is therefore likewisea method for producing a feedstuff or foodstuff, in which a particulatebiomass and/or an aqueous suspension according to the invention is used,and is preferably mixed with further feedstuff or foodstuff ingredients.

In a preferred embodiment of the invention, the particulate biomassand/or the aqueous suspension is used for producing a foodstuff orfeedstuff, in which the biomass and/or the aqueous suspension ispreferably mixed with other foodstuff or feedstuff ingredients and isthen processed to give the foodstuff or feedstuff.

The mixture of biomass and/or aqueous suspension and other foodstuff orfeedstuff ingredients is processed in a preferred embodiment by anextrusion process, in order to obtain portions of foodstuff or feedstuffready for sale. Alternatively, a pelleting method may also be used.

A screw or twin-screw extruder is preferably employed in the extrusionprocess. The extrusion process is preferably carried out at atemperature of 80-220° C., particularly 100-190° C., a pressure of 10-40Bar, and a shaft rotational speed of 100-1000 rpm, particularly 300-700rpm. The residence time of the mixture introduced is preferably 5-30seconds, in particular 10-20 seconds.

In a mode of the extrusion process which is preferred in accordance withthe invention, the process comprises a compacting step and a compressionstep.

It is preferred to intimately mix the components with each other beforecarrying out the extrusion process. This is preferably carried out in adrum equipped with vanes. In this mixing step, a preferred embodimentincludes an injection of steam, in particular so as to bring about theswelling of the starch which is preferably present.

Before being mixed with the biomass and/or aqueous suspension, thefurther foodstuff or feedstuff ingredients are preferably comminuted—ifrequired—so as to ensure that a homogeneous mixture is obtained in themixing step. The comminuting of the further foodstuff or feedstuffingredients may be carried out, for example, using a hammer mill.

A further subject of the current invention is therefore a method offeeding animals, wherein a particulate biomass and/or an aqueoussuspension according to the invention are provided to animals,preferably after mixing the particulate biomass and/or the aqueoussuspension with further feedstuff ingredients, wherein the animals arepreferably selected from poultry, swine, minks, ruminants, in particularfrom calves and beef cattle, sheep, goats, companion animals or animalshold in aquaculture.

Alternatively the biomass and/or aqueous suspension according to theinvention may be used in land applications, in particular as (organic)fertilizer, NPC (nitrogen/phosphorous/potassium source), soil enhancer,plant enhancer and/or composting aid, for producing biogas, forwastewater treatment or as alternative fuel, in particular for cementkilns. It might be further used as part of a fermentation medium forproducing microorganisms, in particular for producing further PUFAscontaining biomass.

A further subject of the current invention is therefore a method forenhancing soil, wherein a particulate biomass and/or an aqueoussuspension according to the invention are strewed on and possibly mixedwith ground, in particular with farmland soil or garden soil.

A further subject of the current invention is therefore also a methodfor fertilizing and/or composting ground, in particular farmland orgarden, wherein a particulate biomass and/or an aqueous suspensionaccording to the invention are strewed on and possibly mixed withground, in particular with farmland soil or garden soil.

A further subject of the current invention is therefore also a methodfor producing biogas, wherein a particulate biomass and/or an aqueoussuspension according to the invention is subjected to microbialdegradation under anaerobic conditions, in particular by making use ofmethanogenic bacteria.

A further subject of the current invention is therefore also a methodfor treatment of wastewater, wherein wastewater is mixed with aparticulate biomass and/or an aqueous suspension according to theinvention.

A further subject of the current invention is therefore also a methodfor producing microorganisms, in particular for producing a PUFAscontaining biomass, wherein a particulate biomass and/or aqueoussuspension according to the invention is used as part of thefermentation medium.

The lipids containing cells according to the invention contain inaverage at least 10 wt.-% of lipids, preferably at least 20 or 30, morepreferably at least 40 or 50 wt.-%, of lipids.

The lipids containing cells according to the invention preferablyfurther contain polyunsaturated fatty acids (PUFAs).

The lipids, and in particular PUFAs, containing cells of the biomass arepreferably microbial cells or plant cells. In a preferred embodiment ofthe invention, the cells are capable of producing the PUFAs due to apolyketide synthase system. The polyketide synthase system may be anendogenous one or, due to genetic engineering, an exogenous one.

Accordingly, “delipidated biomass” according to the invention refers inparticular to the residues of such a PUFAs containing cells comprisingbiomass, in particular as disclosed further below, after having beensubjected to an oil isolation process, in particular as disclosedfurther before.

The plant cells may in particular be selected from cells of the familiesBrassicaceae, Elaeagnaceae and Fabaceae. The cells of the familyBrassicaceae may be selected from the genus Brassica, in particular fromoilseed rape, turnip rape and Indian mustard; the cells of the familyElaeagnaceae may be selected from the genus Elaeagnus, in particularfrom the species Oleae europaea; the cells of the family Fabaceae may beselected from the genus Glycine, in particular from the species Glycinemax.

The microbial organisms which contain a PUFAs containing lipid aredescribed extensively in the prior art. The cells used may, in thiscontext, in particular be cells which already naturally produce PUFAs(polyunsaturated fatty acids); however, they may also be cells which, asthe result of suitable genetic engineering methods or due to randommutagenesis, show an improved production of PUFAs or have been madecapable of producing PUFAs, at all. The production of the PUFAs may beauxotrophic, mixotrophic or heterotrophic.

The biomass preferably comprises cells which produce PUFAsheterotrophically. The cells according to the invention are preferablyselected from algae, fungi, particularly yeasts, bacteria, or protists.The cells are more preferably microbial algae or fungi.

Suitable cells of oil-producing yeasts are, in particular, strains ofYarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus,Trichosporon and Lipomyces.

Suitable cells of oil-producing microalgae and algae-like microorganismsare, in particular, microorganisms selected from the phylumStramenopiles (also called Heterokonta). The microorganisms of thephylum Stramenopiles may in particular be selected from the followinggroups of microorganisms: Hamatores, Proteromonads, Opalines,Developayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids,Oomycetes, Hypochytridiomycetes, Commation, Reticulosphaera,Pelagomonas, Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms,Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes,Raphidophytes, Synurids, Axodines (including Rhizochromulinales,Pedinellales, Dictyochales), Chrysomeridales, Sarcinochrysidales,Hydrurales, Hibberdiales, and Chromulinales. Other preferred groups ofmicroalgae include the members of the green algae and dinoflagellates,including members of the genus Crypthecodiurn.

The biomass according to the invention preferably comprises cells, andpreferably consists essentially of such cells, of the taxonLabyrinthulomycetes (Labyrinthulea, net slime fungi, slime nets), inparticular those from the family of Thraustochytriaceae. The family ofthe Thraustochytriaceae (Thraustochytrids) includes the genera Althomia,Aplanochytrium, Aurantiochytrium, Botryochytrium, Elnia, Japonochytrium,Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium,Thraustochytrium, and Ulkenia. The biomass particularly preferablycomprises cells from the genera Aurantiochytrium, Oblongichytrium,Schizochytrium, or Thraustochytrium, above all from the genusSchizochytrium.

In accordance with the invention, the polyunsaturated fatty acid (PUFA)is preferably a highly-unsaturated fatty acid (HUFA).

The cells present in the biomass are preferably distinguished by thefact that they contain at least 20% by weight, preferably at least 30%by weight, in particular at least 35% by weight, of PUFAs, in each casebased on cell dry matter.

According to the current invention, the term “lipid” includesphospholipids; free fatty acids; esters of fatty acids;triacylglycerols; sterols and sterol esters; carotenoids; xanthophylls(e. g. oxycarotenoids); hydrocarbons; isoprenoid-derived compounds andother lipids known to one of ordinary skill in the art.—The terms“lipid” and “oil” are used interchangeably according to the invention.

In a preferred embodiment, the majority of the lipids in this case ispresent in the form of triglycerides, with preferably at least 50% byweight, in particular at least 75% by weight and, in an especiallypreferred embodiment, at least 90% by weight of the lipids present inthe cell being present in the form of triglycerides.

According to the invention, polyunsaturated fatty acids (PUFAs) areunderstood to mean fatty acids having at least two, particularly atleast three, C—C double bonds. According to the invention,highly-unsaturated fatty acids (HUFAs) are preferred among the PUFAs.According to the invention, HUFAs are understood to mean fatty acidshaving at least four C—C double bonds.

The PUFAs may be present in the cell in free form or in bound form.Examples of the presence in bound form are phospholipids and esters ofthe PUFAs, in particular monoacyl-, diacyl- and triacylglycerides. In apreferred embodiment, the majority of the PUFAs is present in the formof triglycerides, with preferably at least 50% by weight, in particularat least 75% by weight and, in an especially preferred embodiment, atleast 90% by weight of the PUFAs present in the cell being present inthe form of triglycerides.

Preferred PUFAs are omega-3 fatty acids and omega-6 fatty acids, withomega-3 fatty acids being especially preferred. Preferred omega-3 fattyacids here are the eicosapentaenoic acid (EPA, 20:5ω-3), particularlythe (5Z,8Z,11Z,14Z,17Z)-eicosa-5,8,11,14,17-pentaenoic acid, and thedocosahexaenoic acid (DHA, 22:6ω-3), particularly the(4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid.

In a very preferred embodiment of the current invention, cells, inparticular a Schizochytrium strain, is employed which produces asignificant amount of EPA and DHA, simultaneously, wherein DHA ispreferably produced in an amount of at least 20 wt.-%, preferably in anamount of at least 30 wt.-%, in particular in an amount of 30 to 50wt.-%, and EPA is produced in an amount of at least 5 wt.-%, preferablyin an amount of at least 10 wt.-%, in particular in an amount of 10 to20 wt.-% (in relation to the total amount of lipid as contained in thecells, respectively). DHA and EPA producing Schizochytrium strains canbe obtained by consecutive mutagenesis followed by suitable selection ofmutant strains which demonstrate superior EPA and DHA production and aspecific EPA:DHA ratio. Any chemical or nonchemical (e.g. ultraviolet(UV) radiation) agent capable of inducing genetic change to the yeastcell can be used as the mutagen. These agents can be used alone or incombination with one another, and the chemical agents can be used neator with a solvent.

Preferred species of microorganisms of the genus Schizochytrium, whichproduce EPA and DHA simultaneously in significant amounts, as mentionedbefore, are deposited under ATCC Accession No. PTA-10208, PTA-10209,PTA-10210, or PTA-10211, PTA-10212, PTA-10213, PTA-10214, PTA-10215.

The suspension of biomass according to the present invention ispreferably a fermentation broth. The suspension, in particular thefermentation broth, has preferably a biomass density of at least 80 or100 g/l, in particular of 80 or 100 g/l to 250 g/l, preferably at least120 or 140 g/l, in particular 120 g/l or 140 g/l to 220 g/l, morepreferably at least 160 or 180 g/l, in particular 160 g/l to 200 g/l(calculated as dry-matter content). Thus, the suspension may be obtainedby culturing and growing suitable cells in a fermentation medium underconditions whereby the PUFAs are produced by the microorganism.

Methods for producing the biomass, in particular a biomass whichcomprises cells containing lipids, in particular PUFAs, particularly ofthe order Thraustochytriales, are described in detail in the prior art(see e.g. WO91/07498, WO94/08467, WO97/37032, WO97/36996, WO01/54510).As a rule, the production takes place by cells being cultured in afermenter in the presence of a carbon source and of a nitrogen source,along with a number of additional substances like minerals that allowgrowth of the microorganisms and production of the PUFAs. In thiscontext, biomass densities of more than 100 grams per litre andproduction rates of more than 0.5 gram of lipid per litre per hour maybe attained. The process is preferably carried out in what is known as afed-batch process, i.e. the carbon and nitrogen sources are fed inincrementally during the fermentation. When the desired biomass has beenobtained, lipid production may be induced by various measures, forexample by limiting the nitrogen source, the carbon source or the oxygencontent or combinations of these.

In a preferred embodiment of the current invention, the cells are grownuntil they reach a biomass density of at least 80 or 100 g/l, inparticular of 80 or 100 g/l to 250 g/l, preferably at least 120 or 140g/l, in particular 120 g/l or 140 g/l to 220 g/l, more preferably atleast 160 or 180 g/l, in particular 160 g/l to 200 g/l (calculated asdry-matter content). Such processes are for example disclosed in U.S.Pat. No. 7,732,170.

Preferably, the cells are fermented in a medium with low salinity, inparticular so as to avoid corrosion. This can be achieved by usingchlorine-free sodium salts as the sodium source instead of sodiumchloride, such as, for example, sodium sulfate, sodium carbonate, sodiumhydrogen carbonate or soda ash. Preferably, chloride is used in thefermentation in amounts of less than 3 g/l, in particular less than 500mg/l, especially preferably less than 100 mg/l.

Suitable carbon sources are both alcoholic and non-alcoholic carbonsources. Examples of alcoholic carbon sources are methanol, ethanol andisopropanol. Examples of non-alcoholic carbon sources are fructose,glucose, sucrose, molasses, starch and corn syrup.

Suitable nitrogen sources are both inorganic and organic nitrogensources. Examples of inorganic nitrogen sources are nitrates andammonium salts, in particular ammonium sulphate and ammonium hydroxide.Examples of organic nitrogen sources are amino acids, in particularglutamate, and urea.

In addition, inorganic or organic phosphorus compounds and/or knowngrowth-stimulating substances such as, for example, yeast extract orcorn steep liquor, may also be added so as to have a positive effect onthe fermentation.

The cells are preferably fermented at a pH of 3 to 11, in particular 4to 10, and preferably at a temperature of at least 20° C., in particular20 to 40° C., especially preferably at least 30° C. A typicalfermentation process takes up to approximately 100 hours.

After the fermentation has ended, the cells may be pasteurized in orderto kill the cells and to deactivate enzymes which might promote lipiddegradation. The pasteurization is preferably effected by heating thebiomass to a temperature of 50 to 121° C., preferably 50 to 70° C., fora period of 5 to 150 minutes, in particular 20 to 100 minutes.

Likewise, after the fermentation is ended, antioxidants may be added inorder to protect the PUFAs present in the biomass from oxidativedegradation. Preferred antioxidants in this context are BHT, BHA, TBHA,ethoxyquin, beta-carotene, vitamin E, in particular tocopherol, andvitamin C. The antioxidant, if used, is preferably added in an amount of0.001 to 0.1 wt.-%, preferably in an amount of 0.002 to 0.05 wt.-%,relating to the total amount of the fermentation broth after addition ofthe antioxidant.

WORKING EXAMPLES Example 1: Preparation of the Lysed And ConcentratedFermentation Broth

An unwashed cell broth containing microbial cells (Schizochytrium sp.)at a biomass density of over 100 g/l was heated to 60° C. in an agitatedvessel. After heating up the suspension, the pH was adjusted to 7.5 byusing caustic soda (50 wt.-% NaOH solution), before an alcalase(Alcalase® 2.4 FG (Novozymes)) was added in liquid form in an amount of0.5 wt.-% (by weight broth). Stirring was continued for 3 hours at 60°C. After that, the lysed cell mixture was transferred into a forcedcirculation evaporator (obtained from GEA, Germany) and heated to atemperature of 85° C. The mixture was concentrated in the forcedcirculation evaporator, until a total dry matter content of about 35wt.-% was reached.

Example 2: Demulsification Using a Mixture of Hydrophobic Silica andSurfactants

To a sample of 1000 g of the lysed and concentrated fermentation brothas obtained according to example 1 were added 5 g of a demulsifiermixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-%of a hydrophobic silica selected from Sipernat® D10, Sipernat® D17 andAerosil® 202 R (Evonik Industries, Germany).—As negative control 5 g ofthe mixture of Tween 80 and Span 80 was used without addition ofhydrophobic silica.—The demulsifier mixture was prepared by thoroughlymixing the components before applying the mixture. The suspension thusobtained, after addition of the demulsifier mixture, was heated in anagitated vessel to a temperature of 90° C. Samples of 100 ml were takenafter 1 and 22 hours, respectively, and oil was subsequently separatedfrom the aqueous phase by centrifugation. The results are disclosed inthe following table.

TABLE 1 Demulsification using a mixture of hydrophobic silica andsurfactants Hydrophobic Yield after Yield after 22 FFA after 22surfactant one hour hours hours [wt.-%] Sipernat D10 83.5% 86.8% 0.60Sipernat D17 83.2% 92.1% 0.60 Aerosil 202 R 86.3% 87.0% 0.55 none Nofree oil No free oil —As can be seen a high yield of oil can be obtained already after onehour of incubation, but the yield is higher after 22 hours ofincubation.

Example 3: Demulsification Using Different Amounts of DemulsifierMixture

To samples of 1000 g of the lysed and concentrated fermentation broth asobtained according to example 1 were added 1, 3, 5 or 8 g of ademulsifyer mixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80and 5 wt.-% Sipernat® D17 (Evonik Industries, Germany), corresponding toa final amount of hydrophobic silica in the suspension of 0.005, 0.015,0.025 and 0.040 wt.-%, respectively.

The suspensions thus obtained were heated in an agitated vessel to atemperature of 90° C. Samples of 100 ml were taken after 24 hours,respectively, and oil was subsequently separated from the aqueous phaseby centrifugation.

The results are disclosed in the following table.

TABLE 2 Demulsification using a mixture of hydrophobic silica andsurfactants Amount of Amount of hydrophobic demulsifyer silica in aqeousYield after FFA after 24 mixture suspension 24 hours hours [wt.-%] 0.1wt.-% 0.005 wt.-% 87.4% 0.60 0.3 wt.-% 0.015 wt.-% 87.1% 0.70 0.5 wt.-%0.025 wt.-% 89.0% 0.70 0.8 wt.-% 0.040 wt.-% 86.6% 0.75As can be seen, an efficient demulsification takes place already byadding an amount of demulsifier mixture of 1 g per kg suspension,corresponding to an amount of hydrophobic silica in the suspension of0.005 wt.-%.

Example 4: Demulsification at Different Incubation Times

To a sample of 1000 g of the lysed and concentrated fermentation brothas obtained according to example 1 were added 5 g of a demulsifiermixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-%Sipernat® D17 (Evonik Industries, Germany), corresponding to a finalamount of hydrophobic silica in the suspension of 0.025 wt.-%.

The suspension thus obtained was heated in an agitated vessel to atemperature of 90° C. Samples of 100 ml were taken after 2, 4, 6 and 24hours, respectively, and oil was subsequently separated from the aqueousphase by centrifugation.

The results are disclosed in the following table.

TABLE 3 Demulsification using a mixture of hydrophobic silica andsurfactants Incubation time [hours] Yield FFA [wt.-%] 2 86.7% 0.36 484.6% 0.38 6 88.4% 0.41 24 89.0% 0.70As can be seen, and in accordance with the results as disclosed in table1, an efficient demulsification takes already place with shortincubation times, but better yields are achieved with incubation timesof more than 5 hours.—The advantage of relatively short incubation timesis the very low amount of free fatty acids (FFA) in the final product.

Example 5: Demulsification at Different Temperatures

To four samples of 1000 g of the lysed and concentrated fermentationbroth as obtained according to example 1 were added 5 g of a demulsifiermixture containing 47.5 wt.-% Tween 80, 47.5 wt.-% Span 80 and 5 wt.-%Sipernat® D17 (Evonik Industries, Germany), respectively, correspondingto a final amount of hydrophobic silica in the suspensions of 0.025wt.-%.

The samples such obtained were heated in agitated vessels for 24 hoursat different temperatures of 30, 50, 70 and 90° C., respectively.Samples of 100 ml were taken after 24 hours, respectively, and oil wassubsequently separated from the aqueous phase by centrifugation.

The results are disclosed in the following table.

TABLE 4 Demulsification at different temperatures Incubation temperature[° C.] Yield FFA [wt.-%] 30 77.8% 0.37 50 72.0% 0.38 70 85.0% 0.40 9089.0% 0.70As can be seen, demulsification takes place already at very lowtemperatures, but good yields are obtained at temperatures above 50°C.—Like for low incubation times, lower FFA values are also obtained atincubation at lower temperatures.

Example 6: Variation of the Amount and Nature of Surfactants in theDemulsifier Mixture

To samples of 1000 g of the lysed and concentrated fermentation broth asobtained according to example 1 were added different amounts of ademulsifier mixture containing the hydrophobic silica Sipernat® D17(Evonik Industries, Germany) and optionally different amounts ofsurfactants.

The samples such obtained were heated in agitated vessels at 90° C.Samples of 100 ml were taken after 24 hours, respectively, and oil wassubsequently separated from the aqueous phase by centrifugation.

The different compositions of the demulsifier mixture and amounts asadded are disclosed in the following table, as well as the yields of oilas obtained:

TABLE 5 Composition and employed amounts of demulsifier mixtures Comp.Surfactant 1 Surfactant 2 Hydrophobic silica No. [wt.-%] [wt.-%] [wt.-%]1 — — 100 Sipernat D17 2 45.5 Tween 80 45.5 Span 80 9.0 Sipernat D17 348.4 Tween 80 48.4 Span 80 3.2 Sipernat D17 4 47.5 Tween 80 47.5 Span 805.0 Sipernat D17 5 71.4 Tween 80 23.8 Span 80 4.8 Sipernat D17 6 23.8Tween 80 71.4 Span 80 4.8 Sipernat D17 7 47.6 Brij 98 47.6 Brij 92 4.8Sipernat D17

TABLE 6 Influence of surfactants on the oil yield Comp. Amount Amount ofhydrophobic FFA No. added [g] silica in aq. suspension Yield [wt.-%] 10.24 0.024 wt.-% 84.2% 0.70 2 2.5 0.023 wt.-% 86.4% 0.75 3 7.5 0.024wt.-% 90.2% 0.75 4 5 0.025 wt.-% 89.0% 0.70 5 5 0.024 wt.-% 88.6% 0.70 65 0.024 wt.-% 86.5% 0.75 7 5 0.024 wt.-% 87.7% 0.60As can be seen, efficient demulsification does take place with differentsurfactants and even without using surfactants. But surfactantsobviously have a positive effect on the yield.

The invention claimed is:
 1. A method of isolating lipids from a lipidscontaining biomass, comprising the following steps: a) providing abiomass suspension, wherein the biomass comprises cells containing anaverage of at least 10 wt.-% of lipids; b) adding hydrophobic silica tothe suspension; c) heating the suspension thus obtained to a temperatureof at least 50° C. and then incubating the suspension for at least 20minutes; d) separating an oil containing light phase from a water,salts, cell debris and residual oil containing aqueous phase.
 2. Themethod of claim 1, wherein the hydrophobic silica is added to thesuspension to a final concentration of between 0.005 and 0.25 wt.-%. 3.The method of claim 2, wherein the hydrophobic silica is added to thesuspension so as to adjust the final concentration to be between 0.015to 0.10 wt.-% of hydrophobic silica.
 4. The method of claim 1, whereinbesides silica, at least one surfactant is added to the suspension to afinal concentration of surfactant of between 0.1 and 5.0 wt.-%.
 5. Themethod of claim 4, wherein besides silica, at least one surfactant isadded to the suspension in an amount so as to adjust the finalconcentration of surfactant to be between 0.25 and 3.0 wt.-%.
 6. Themethod of claim 1, wherein the biomass suspension of step a) has a totaldry matter content of between 20 and 60 wt.-%.
 7. The method of claim 6,wherein hydrophobic silica is at a final concentration of between 0.005and 0.25 wt.-%, and surfactant is at a final concentration of between0.1 and 5.0 wt.-%.
 8. The method of claim 1, wherein the suspension isheated in step c) to a temperature of 50 to 100° C.
 9. The method ofclaim 8, wherein the suspension is heated in step c) to a temperature of65 to 90° C.
 10. The method of claim 1, wherein the suspension isincubated in step c) for 20 minutes to 30 hours.
 11. The method of claim1, wherein the suspension is incubated in step c) for one hour to 24hours.
 12. The method of claim 1, further comprising, as an additionalstep, lysing the cells of the biomass, wherein lysing is carried outbefore adding hydrophobic silica.
 13. The method of claim 1, wherein thebiomass suspension is a fermentation broth and the cells are of thegenus Schizochytrium.
 14. Oil comprising hydrophobic silica in an amountof 5 ppm (w/w) to 10 wt.-%, and wherein the oil comprises PUFAs in anamount of at least 10 wt.-%.
 15. The oil of claim 14, wherein the oilcomprises hydrophobic silica in an amount of 0.5 wt.-% to 2.5 wt.-%, andwherein the oil comprises PUFAs in an amount of 45 to 60 wt.-%.
 16. Theoil of claim 14, wherein the oil comprises fatty acid esters, in anamount of at least 50 wt.-%, and free fatty acids (FFA) in an amount ofless than 1 wt.-%.
 17. The oil of claim 16, wherein the oil comprisesfatty acid esters, in an amount of at least 85 wt.-%, and free fattyacids (FFA) in an amount of less 0.8 wt.-%.
 18. A PUFAs containingdelipidated biomass comprising: 10 to 25 wt.-% of ashes; less than 0.1wt.-% of a non-polar organic solvent; and less than 0.1 wt.-%, ofchloride; wherein the biomass contains cells and/or cell debris of thegenus Schizochytrium.
 19. The PUFAs containing delipidated biomass ofclaim 18, wherein the PUFAs containing delipidated biomass comprises15-20 wt.-% of ashes, less than 0.05 wt.-%, of a non-polar organicsolvent and less than less than 0.05 wt.-%, of chloride.
 20. The PUFAscontaining delipidated biomass of claim 4, wherein the biomass comprisesa mixture of DHA and EPA in a ratio of about 3:2 to about 4:1, andwherein the content of DHA is at least 10 wt.-% of the total amount oflipid.